Modulation of mAb quality attributes using microliter scale fed‐batch cultures

Abstract

A high-throughput DoE approach performed in a 96-deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO-S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed-batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N-glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines - some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small-scale bioreactors. Being fast and resource effective, this integrated high-throughput approach can provide information which is particularly useful during early stage cell culture development.

Tailoring recombinant protein quality by rational media design

“…The important preceding optimization efforts of the proprietary fed-batch process platform enable to maintain high viabilities, in general greater than 80% at the end of the culture of a 14-day run 19,25,251 . As a result the current cell-culture platform process delivers consistently high titers and rather low aggregate and lowmolecular-weight species.…”

“…Cell culture media design has a great potential to modulate the key quality attributes of the molecule 25 , and in particular by means of high-throughput design of experiment (DoE) approaches 26,27 , due to its central role in the upstream manufacturing 19 . The environment the cell is cultured in closely influences its metabolism to a great extent [20][21][22] .…”

“…The modulation of the glycosylation level can be obtained through media complementation with precursors and/or cofactors, aiming to reduce the formation of ammonium by limiting the amino acid concentrations in the medium 25 . It has also been shown that increased substrate levels, in particular, nucleotide-sugar precursors including UDP-Hex, UDP-HexNAc, and CMP-sialic acid induced an overall increase of the glycosylation flux 86 .…”

“…The adjustment of the levels of a choice of medium components and the addition of novel compounds either in the medium or the in bolus feeds, influence the enzymatic reactions in the endoplasmic reticulum and in the Golgi apparatus, the substrate generation as well as their transport into the two organelles 23,24 . Further enhancements and technology advancements of high-throughput cell culture systems, including 96-deepwell plates and TubeSpin bioreactor tubes, allow to test a plethora of different supplements at various concentrations simultaneously [25][26][27] . Moreover, the observed trends in these systems may be reproduced in controlled bioreactor systems, including lab-scale bioreactors 28 , which highlights the great value of small-scale high-throughput compound screening for the identification of new levers that tailor recombinant protein quality attributes.…”

“…Supplementation was assessed in high-throughput systems including 96-deepwell plates and shake tubes. 96-deepwell plates have proven to be a reliable system to screen raw materials impacting product quality attributes of recombinant proteins 25,27,28 . Confirmation of the findings was conducted in lab-scale 3.5-liter bioreactors.…”